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    TC AP3

    Uploaded by

    Silvia Borges

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    © All Rights Reserved
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    TC AP3

    Uploaded by

    Silvia Borges
    15 views21 pages

    Original Title

    TcAP3

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    15 views21 pages

    TC AP3

    Uploaded by

    Silvia Borges

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    © All Rights Reserved
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    of 21

    Técnicas Histoenzimáticas - Princípios Teóricos

    Miofosforilase

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    Solutions:
    VI. Ethanol, 95 % (v/v)
    I. Glucose-1-Phosphate, 1% (w/v)
    Glucose-1-phosphate 500 mg
    VII. Lugol's Iodine Solution: (store at
    room temp. in an amber bottle)
    II. Adenosine Monophosphate, 0.2% (w/v)
    Potassium Iodide (KI) 1 g
    Adenosine monophosphate 100 mg
    Dissolve in deionized water 1
    ml When completely
    III. Sodium Acetate Solution, 0.1 M
    dissolved add iodine crystals
    Sodium acetate CH3COONa3H2O
    (I2) 0.5 g
    2.72 g
    When iodine is completely
    Deionized water 200 ml
    dissolved add D.I. H2O to a
    final volume 25 ml
    IV. Acetic Acid, 0.1N
    CH3COOH 0.575 ml
    VIII. Working Solution for Stain
    Add to deionized water 100 ml
    (prepare fresh for each assay)
    Thaw ahead and bring to
    IV. Sodium Acetate Buffer, pH 5.6, 0.1 M
    room temperature
    Solution III: 181 ml
    To a 20 ml glass beaker add
    Solution IV: 19 ml
    solutions I, II, V
    Add 1.5 ml 100 %(absolute)
    V. Glycogen, 0.02%
    Ethanol
    Glycogen 20 mg
    Adjust pH to 5.6
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    Staining Procedure:

    1. Place coverslips with sections in a columbia staining dish


    (Thomas Scientific #8542-C12) for 5 minutes at room temperature
    for a minimum of 45 minutes (overnight is very acceptable).

    2. Wash with several exchanges of deionized H2O.

    3. Add 6 - 8 drops of Lugol's Iodine to approx. 10 ml deionized


    water.

    4. Add iodine solution to sections until they are dark in color.


    5. Wipe the back of the coverslip with a cotton tipped swab to
    remove excess iodine solution.

    6. Mount coverslip onto a labeled glass slide with glycerol that


    has been colored with a few drops of Lugol's Iodine.

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    PRINCIPLE:

    - In the histochemical reaction, phosphorylase acts on the substrate, glucose-1-


    phosphate and forms, in the presence of a glycogen primer, a polysaccharide
    composed of -1,4-glycosyl units.

    - The in vitro reaction of polysaccharide formation is, therefore, the opposite of


    the in vivo action of glycogen degradation.

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    - The concentration of the substrate is high, and the concentration of the
    inorganic phosphate is low.

    - The system equilibrium therefore favors glycogen formation.

    - Adenosine-5'-monophosphate functions as an activator.

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    - Exposure of the sections to an iodine solution after incubation results
    in a varied color formation in the newly formed polysaccharide

    - A negative reaction is yellow, and it has been shown that unbranched chains of 4
    to 6 glucosyl units will give a negative reaction.

    - A polysaccharide of 8 to 12 units gives a reddish color, followed by various


    transitional colors as the length of the chain increases.

    - Chain lengths of 30 to 35 units give a blue color

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    - A negative reaction is yellow, and it has been shown that unbranched chains of 4
    to 6 glucosyl units will give a negative reaction.

    - A polysaccharide of 8 to 12 units gives a reddish color, followed by various


    transitional colors as the length of the chain increases.

    - Chain lengths of 30 to 35 units give a blue color

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